First reported case of Doyne honeycomb retinal dystrophy (Malattia Leventinese/autosomal dominant drusen) in Scandinavia.

BACKGROUND: Doyne honeycomb retinal dystrophy (DHRD)/malattia leventinese (ML) is an autosomal dominant, progressive retinal disorder characterized by massive central retinal drusen often partly coalescent forming a characteristic honeycomb-like pattern. Debut of vision loss often occurs in early to mid-adulthood, and the degree varies. A single variant in EFEMP1: c.1033C>T (R345W) has been identified as the cause in all cases. METHODS: Following DNA isolation, exome sequencing was performed in seven genes associated with flecked retina. Direct sequencing was used for variant verification. RESULTS: We report the first Scandinavian case of molecular genetically verified DHRD/ML: a 57-year-old woman debuting with vision loss and metamorphopsia. On both eyes, ophthalmological findings included massive hard drusen in the macular region and nasal to the optic disc as well as macular hyperpigmentation. Secondary choroidal neovascularizations were identified on both eyes, and anti-vascular endothelial growth factor was administered, without effect. CONCLUSION: Molecular genetic investigation revealed heterozygosity for the known pathogenic missense variant in EFEMP1: c.1033C>T (R345W) previously reported in relation to DHRD/ML. Family history revealed no other cases of similar visual impairment suggesting a de novo mutation. Furthermore, there was no correlation between the unique DHRD/ML haplotypes reported in the literature and our patient.

Fibulin-3 knockout mice demonstrate corneal dysfunction but maintain normal retinal integrity.

Fibulin-3 (F3) is an extracellular matrix glycoprotein found in basement membranes across the body. An autosomal dominant R345W mutation in F3 causes a macular dystrophy resembling dry age-related macular degeneration (AMD), whereas genetic removal of wild-type (WT) F3 protects mice from sub-retinal pigment epithelium (RPE) deposit formation. These observations suggest that F3 is a protein which can regulate pathogenic sub-RPE deposit formation in the eye. Yet the precise role of WT F3 within the eye is still largely unknown. We found that F3 is expressed throughout the mouse eye (cornea, trabecular meshwork (TM) ring, neural retina, RPE/choroid, and optic nerve). We next performed a thorough structural and functional characterization of each of these tissues in WT and homozygous (F3-/-) knockout mice. The corneal stroma in F3-/- mice progressively thins beginning at 2 months, and the development of corneal opacity and vascularization starts at 9 months, which worsens with age. However, in all other tissues (TM, neural retina, RPE, and optic nerve), gross structural anatomy and functionality were similar across WT and F3-/- mice when evaluated using SD-OCT, histological analyses, electron microscopy, scotopic electroretinogram, optokinetic response, and axonal anterograde transport. The lack of noticeable retinal abnormalities in F3-/- mice was confirmed in a human patient with biallelic loss-of-function mutations in F3. These data suggest that (i) F3 is important for maintaining the structural integrity of the cornea, (ii) absence of F3 does not affect the structure or function of any other ocular tissue in which it is expressed, and (iii) targeted silencing of F3 in the retina and/or RPE will likely be well-tolerated, serving as a safe therapeutic strategy for reducing sub-RPE deposit formation in disease. KEY MESSAGES: • Fibulins are expressed throughout the body at varying levels. • Fibulin-3 has a tissue-specific pattern of expression within the eye. • Lack of fibulin-3 leads to structural deformities in the cornea. • The retina and RPE remain structurally and functionally healthy in the absence of fibulin-3 in both mice and humans.

Doyne honeycomb retinal dystrophy/malattia leventinese induced by EFEMP1 mutation in a Chinese family.

BACKGROUND: Doyne honeycomb retinal dystrophy (DHRD)/malattia leventinese (ML) is a rare allelic condition with massive drusen in the posterior fundus caused by EFEMP1 gene mutation. Patients showed decreased vision when the lesion affected the macular area. At present, the treatment efficiency is not satisfactory. CASE PRESENTATION: In this study, we presented a family with DHRD/ML disease and analyzed the pathological and genetic information. A 28-year-old female patient presented to our department due to impaired visual acuity for 10 years especially in the right eye with deterioration for 5 months. Gene sequencing was performed by MyGenostics (Peking, China). Gene sequencing results revealed heterozygous mutations in EFEMP1 gene, which were consistent with the DHRD/ ML. Single heterozygous mutation (c.1033C > T) was observed in each of the three blood samples. This missense mutation triggered p.R345W. CONCLUSIONS: DHRD/ML is a rare disease associated with EFEMP1 gene mutation. Up to now, we are not sure whether these lesions are associated with the onset of DHRD/ML. In future, we hope to find out the exact relationship between them.

Mapping wild-type and R345W fibulin-3 intracellular interactomes.

Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate 'heavy' and 'light' isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.

Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy: Similarities to Age-Related Macular Degeneration and Potential Therapies.

Fibulin-3 (F3) is a secreted, disulfide-rich glycoprotein which is expressed in a variety of tissues within the body, including the retina. An Arg345Trp (R345W) mutation in F3 was identified as the cause of a rare retinal dystrophy, Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD). ML/DHRD shares many phenotypic similarities with age-related macular degeneration (AMD). The most prominent feature of ML/DHRD is the development of radial or honeycomb patterns of drusen which can develop as early as adolescence. Two independent mouse models of ML/DHRD show evidence of complement activation as well as retinal pigment epithelium (RPE) atrophy, strengthening the phenotypic connection with AMD. Because of its similarities with AMD, ML/DHRD is receiving increasing interest as a potential surrogate disease to study the underpinnings of AMD. This mini-review summarizes the current knowledge of F3 and points toward potential therapeutic strategies which directly or indirectly target cellular dysfunction associated with R345W F3.

Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration.

An R345W mutation in the N-glycoprotein, fibulin-3 (F3), results in inefficient F3 folding/secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N-Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N-glycosylation alters the protein homeostasis or proteostasis of wild-type (WT) and R345W F3 in ARPE-19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N-glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% (vs. WT F3). Genetic elimination of F3 N-glycosylation (via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum (ER) chaperones GRP78 (glucose-regulated protein 78) and GRP94 (glucose-regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/R345W F3 interacted with GRP94, while substantially less N249Q and N249Q/R345W interacted with the ER lectins than their N-glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/R345W F3 secretion (by 62%), demonstrating this variant's unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N-glycosylation to acquire a stable, native-like structure.

Differential tolerance of 'pseudo-pathogenic' tryptophan residues in calcium-binding EGF domains of short fibulin proteins.

An Arg345Trp (R345W) mutation in the last canonical calcium-binding epidermal growth factor (cbEGF) domain of fibulin-3 (F3) causes the rare macular dystrophy, Malattia Leventinese (ML). In cell culture studies, this mutation leads to inefficient F3 secretion and higher intracellular steady state levels, likely due to F3 disulfide bonding and/or protein folding problems. However, how the R345W mutation actually causes ML is still largely unknown. Herein we tested whether the introduction of analogous, 'pseudo-pathogenic' tryptophan mutations immediately after the bn cysteine (bn+1) in other cbEGF domains also caused protein folding/secretion challenges. We found that introduction of tryptophan mutations into each of the four other F3 canonical cbEGF domains caused a significant reduction in protein secretion ranging from 2.7 to 56% of wild-type (WT) F3 levels. Surprisingly, an R185W mutation in the first canonical cbEGF domain of F3 yielded the highest amount of secretion among the F3 tryptophan mutants, and its secretion defect could be rescued to near WT levels (95%) after growth temperature reduction. Interestingly, when similarly positioned tryptophan mutations were introduced into any of the canonical cbEGF domains of the highly homologous protein, fibulin-5 (F5), there was no effect on secretion. In an attempt to make F3 tolerant of tryptophan residues (like F5), we genetically engineered F3 to have a higher sequence homology with F5 by deleting three insert regions present in F3, but not F5. However, deletion of one or more of these regions did not have a beneficial effect on R345W F3 secretion. Overall, these results demonstrate that the introduction of tryptophan residues at the bn+1 position does not universally disrupt cbEGF domain folding and secretion, but that their effect is context dependent, and in this case, uniquely disrupt the folding of canonical cbEGF domains of F3, but not F5.

A high-throughput cell-based Gaussia luciferase reporter assay for identifying modulators of fibulin-3 secretion.

An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3-related diseases.